The present invention relates to nucleic acid sequences which comprise all or part of a nucleic acid sequence of a CIITA gene. These sequences can comprise a sequence which exhibits a transcriptional promoter activity, which activity is, in particular, specifically expressed in one cell type. The sequences can also comprise a coding sequence.Therapeutic and diagnostic applications, in particular relating to disorders in which it is desirable to act on the level at which genes which encode the class II molecules of the major histocompatibility complex (MHC) are expressed.

Description

29 parts

›This application claims priority under 35 U.S.C. §§119…

This application claims priority under 35 U.S.C. §§119 and/or 365 to Patent Application No. 97-04954 filed in France on Apr. 22, 1997; the entire content of which is hereby incorporated by reference.

The present invention relates to novel nucleic acid sequences which can be involved in controlling and regulating the expression of genes encoding MHC type II molecules and to their use, in particular as drugs for treating disorders in which it is desirable to act on the level at which genes encoding MHC type II molecules are expressed.

The molecules of the class II major histo-compatibility complex (termed MHC in that which follows) are heterodimeric transmembrane glycoproteins which are directly involved in activating T helper CD4+ lymphocytes during the course of the immune response.

In man, this class II complex is represented by the molecules which belong to the HLA (human leucocyte antigen) system. The genes which encode the α and β chains of which the HLA-DR, HLA-DQ and HLA-DP molecules are composed are located within the D region of chromosome 6.

Expression of these genes is very highly regulated. In contrast to the genes which encode the MHC type I molecules, which are expressed ubiquitously, expression of the genes which encode the MHC class II molecules is either constitutive, in only a few cell types such as B lymphocytes, activated T lymphocytes, macrophages, cells of the thymic epithelium, or dendritic cells such as the Langerhans cells, or is induced following stimulation, for example by cytokines, more specifically by interferon γ (INF γ) or interleukin 4 (IL4), in several other cell types such as cells which belong to the macrophage or monocyte line, endothelial cells, fibroblasts, muscle cells or cancer cells such as melanoma cells.

Furthermore, expression of the genes which encode MHC class II molecules in B lymphocytes is transient. Thus, differentiation of the B cells into plasma cells which produce the immunoglobulins is accompanied by the suppression of certain genes including those which encode MHC class II.

Similarly, it has been shown that the level at which MHC type II molecules are expressed is a determining factor in the process of T cell activation.

As a consequence, it is clearly apparent that the molecular mechanisms by which expression of these genes is regulated constitute a key element in the efficacy of the immune response. Any defect in this regulatory process may result in significant immunological disorders or autoimmune diseases. Thus, abnormal expression of the MHC class II genes has in some cases been observed at the surface of cells which should not normally express these genes. Similarly, it is possible to observe over-expression of these genes, leading to an activation of the CD4+ lymphocytes which is aberrant and uncontrolled [Bottazzo et al., 1986, Immunol. Rev., 94, 137-169]. Events of this kind could, at least in part, be responsible for disorders such as insulin-dependent diabetes, multiple sclerosis, rheumatoid arthritis and lupus erythematosus. Conversely, it has been possible to demonstrate an immunodeficiency in some patients which has resulted from a disturbance in the expression of MHC class II genes. Mention may, for example, be made of the BLS (bare lymphocytes syndrome) syndrome which is a recessive autosomal disorder in which expression of the MHC class II genes is very limited if not to say non-existent, a situation which finds expression in the absence of cellular and humoral immune responses and is accompanied by a large number of infections which are often fatal.

Several scientific groups have analysed the mechanisms by which expression of the MHC class II genes is regulated and have identified a number of transactivating molecules which are capable of binding, directly or indirectly, to promoter sequences which are specific for the said genes [for a review, see Mach et al., 1996, Annu. Rev. Immunol. 14, 301-331].

The applicant has previously identified and characterized one of these factors, i.e. the CIITA factor (class II transactivator) [Steimle et al., 1993, Cell 75, 135-146 and EP 648836]. Furthermore, document WO 9606107 shows that there are two domains within the CIITA factor which are more involved in activating transcription of the MHC class II genes, more specifically the domain which is defined by SEQ ID No. 21 of the present invention and which corresponds to the translation of the nucleic acid sequence according to SEQ ID No. 17. Nevertheless, surprisingly and contrary to that which is observed in the case of other factors which are involved in regulating expression of the MHC class II genes (Cogswell et al., 1991, Crit. Rev. Immunol. 11, 87-112), Steimle et al. have demonstrated that expression of the CIITA factor coincides strictly with expression of the MHC class II genes and is required absolutely both for constitutively expressing and for inducing the said MHC genes. Furthermore, Silacci et al. (1994, J. Exp. Med., 180, 1329-1336) have demonstrated that suppression of the MHC class II genes during plasma cell differentiation is associated with suppression of the gene which encodes CIITA factor.

Moreover, Lennon et al. (1997, Immunogenetics, 45, 266-273) have identified the promoter sequence of a CIITA gene, which sequence is responsible for the differential expression of this factor in B cells. However, the existence of this sequence alone does not explain why differential expression of the CIITA factor is observed in different cell types. Furthermore, it does not account for induction by cytokines.

Using samples derived from different tissues of human origin, the applicant has now identified the complex organization of the sequences which ensure regulation of the expression of the CIITA factor, has isolated and characterized other promoter regions and has demonstrated the existence of several forms of CIITA factor, and has also demonstrated the existence of different CIITA genes.

The expression “CIITA gene” is understood as meaning a nucleic acid sequence which consists of a promoter (P) moiety, an untranslated (UT) moiety and a coding (Prot) moiety, with the coding moiety encoding one of the identified forms of CIITA factor.

›More precisely, the inventors have identified a number…

More precisely, the inventors have identified a number of nucleic acid sequences which represent CIITA genes and which are therefore capable, in particular, of being involved in controlling and regulating the expression of genes encoding MHC class II molecules. The expression “nucleic acid sequence which represents CIITA genes” is understood as meaning that the sequence in question comprises all or part of a nucleic acid sequence corresponding to the mRNAs which derive from the different tissues or cell lines which express CIITA activity either constitutively or following induction. Such sequences can therefore equally well be sequences which are at least partially coding, as for example sequences which are involved in controlling the expression, in particular, of sequences which possess a transcriptional promoter activity.

The expression “nucleic acid sequence” is understood as meaning a natural, isolated, or synthetic, double-stranded or single-stranded DNA and/or RNA fragment which designates a precise linked-up series of modified or unmodified nucleotides and which makes it possible to define a fragment or a region of a nucleic acid.

The expression “polypeptide” is understood as meaning a precise, natural, isolated, or synthesized, modified or unmodified linked-up series of amino acids, independently of its size or its function.

The expression “allelic variant” of a polypeptide is understood as meaning the entirety of the mutated polypeptides and the polymorphisms which can exist in man, and which are obtained, in particular, by truncating, substituting, deleting or adding on amino acid residues, as well as the artificial variants which are employed in vitro.

The expression “nucleic acid sequence which exhibits a transcriptional promoter activity” is understood as meaning a nucleic acid sequence which makes it possible to control, that is initiate and/or modulate, the transcription of at least one homologous or heterologous gene which is located downstream of the said sequence. Similarly, reference will be made to the promoter function of the said sequences.

The expression “nucleic acid sequence which is homologous to a first nucleic acid sequence” is understood as meaning a nucleic acid sequence which naturally exhibits a functional link with the said first sequence. Thus, according to the invention, a nucleic acid sequence which exhibits a CIITA promoter activity, that is which naturally directs the transcription of a nucleic acid sequence encoding a CIITA factor, is, for example, considered as being homologous to this same nucleic acid sequence which encodes a CIITA gene. In the opposite case, reference will be made to a “heterologous nucleic acid sequence”.

The expression “reporter gene” is understood as meaning any nucleic acid sequence which is located downstream of a second nucleic acid sequence and which makes it possible to analyse the transcriptional promoter activity of the said second sequence. Thus, transcription of this reporter gene is manifested by the appearance of a product (RNA or polypeptide) which can readily be detected using well known conventional techniques.

It should be understood that the present invention does not relate to genomic nucleotide sequences in their natural chromosomal environment, that is in the natural state; on the contrary, the sequences are sequences which have been isolated, meaning that they have been directly or indirectly withdrawn, for example by copying (cDNA), and that their environment has been at least partially modified.

The invention thus relates to a nucleic acid sequence which comprises all or part of a nucleic acid sequence of a CIITA gene and which is selected from the sequences SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, and their complementary sequences.

The invention more specifically relates to such a nucleic acid sequence which comprises all or part of a sequence which exhibits a transcriptional promoter activity.

Particularly interesting sequences which may be mentioned are those which comprise all or part of a sequence which is selected from SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, and their complementary sequences.

The sequences which contain all or part of the sequence identified as SEQ ID No. 4, or its complementary sequence, are particularly advantageous in that they are able to express their transcriptional promoter activity specifically in one cell type, such as dendritic cells.

Certain sequences which have been identified in accordance with the invention are able to express their transcriptional promoter activity following induction by a cytokine such as, for example, interferon γ or interleukin 4. A preferred example of such a sequence is represented by the sequence which comprises all or part of an identified sequence SEQ ID No. 6, or its complementary sequence.

The invention also relates to the nucleic acid sequences which comprise all or part of a sequence selected from:

a) a nucleic acid sequence which encodes a polypeptide which possesses an amino acid sequence such as shown in SEQ ID No. 16, and its complementary sequence,

b) the sequences SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 10 and SEQ ID No. 11 and their complementary sequences,

c) a nucleic. acid sequence which encodes an allelic variant of a polypeptide such as defined in a), or its complementary sequence.

The present invention furthermore relates to a nucleic acid sequence which comprises at least one sequence exhibiting a transcriptional promoter activity, such as, in particular, the sequences which comprise all or part of the sequences SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6, or their complementary sequences, which sequence is located upstream of at least one heterologous or homologous nucleic acid sequence such as, for example, a nucleic acid sequence which comprises all or part of a sequence selected from:

a) a nucleic acid sequence which encodes a polypeptide which consists of the amino acids identified in SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18 or SEQ ID No. 19, or its complementary sequence,

›b) the identified nucleic acid sequences is SEQ…

b) the identified nucleic acid sequences is SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14 and SEQ ID No. 15, and their complementary sequences,

c) a nucleic acid sequence which encodes an allelic variant of a polypeptide such as defined in a), or its complementary sequence.

It should be made clear that, in this case, it is possible to have at least two sequences which exhibit a transcriptional promoter activity and/or at least two heterologous or homologous nucleic acid sequences which are situated contiguously or at a distance in relation to each other, and in the same direction or in opposite directions, without the transcriptional promoter function or the transcription of the said sequences thereby being affected.

Similarly, it is possible, in this type of nucleic acid construction, to introduce “neutral” nucleic acid sequences or introns which do not affect transcription and which are spliced before the translation step. Sequences of this nature, and their uses, are widely described in the literature.

According to the invention, the nucleic acid sequences, or their fragments, can, in particular, encode all or part of polypeptides which possess the amino acid sequence of a CIITA factor as described in the present invention.

It will then be stated that they encode CIITA polypeptides.

The sequences can also be employed as probes or as primers in processes for detecting or identifying or enzymically amplifying nucleic acid. In this case, the fragments exhibit a minimum size of 10 bases, and preference will be given to fragments of 20 bases, preferably of 30 bases.

The present invention also relates to a nucleic acid sequence which possesses a sequence which is complementary to a target sequence which belongs to a gene or to an RNA whose expression it is desired to block specifically. An antisense oligonucleotide which hybridizes with the sequence to which it is complementary and can thereby block expression of the mRNA having this sequence constitutes such a sequence. In this context, the term “oligonucleotide” is used in a general manner to designate a polynucleotide of from 2 to 100, more generally of from 5 to 50, ribonucleotides, deoxyribonucloetides or mixed nucleotides in a series. According to the invention, such a sequence is able to hybridize with a nucleic acid sequence which comprises a sequence exhibiting a transcriptional promoter activity or with a nucleic acid sequence comprising a sequence such as previously defined in a), b) or c), and is furthermore able either to block the promoter activity of the said sequence or to inhibit the synthesis of the polypeptide which is encoded by the said sequence.

The hybridization conditions are determined, according to the invention, in order to ensure at least 95% homology. The skilled person is in possession of sufficient knowledge to enable him to define the said conditions.

Even if the described sequences are generally normal sequences, the invention also relates to sequences which are mutated to the extent that they include at least one point mutation and preferably less than 20 mutations in all.

Preferably, the present invention relates to nucleotide sequences in which the point mutations are not silent, that is they lead either to a change in the regulation of the efficacy or of the cellular specificity of the transcription of the gene which is located downstream of the said sequence, or to a change in the coding sequence which affects expression of the CIITA gene, or to a change in the encoded amino acid as compared with the normal sequence, which change affects the function of the corresponding CIITA factor.

The present invention relates, in particular, to a nucleic acid sequence which comprises at least one mutation which affects the transcriptional promoter function of the said sequence. Preferably, these mutations concern the regions which are involved in the transcriptional promoter activity and which make it possible to bind factors which are involved in the transcription initiation, activation or modulation step or in transcription more generally. These regions can, for example, consist of at least one site which is involved in the transcription process and which is selected from the group consisting of the NF-GMb site (Shannon et al., 1988, Proc. Natl. Acad. Sci. USA, 85, 674-678), the NF-IL6 site (Akira and Kishimoto, 1992, Immunil. Rev. 127, 25-50), the PEA3 site (Wasylyk et al., 1989, EMBO J., 8, 3371-3378), the API site (Pollock and Treisman, 1990, Nucleic Acid Res. 18, 6197-6204), the CCAAT box (Dorn et al., 1987, Cell, 50, 863-872), the E2A box (Murre et al., 1989, Cell, 56, 777-783), the IRF1/2 site (Tanaka et al., 1993, Molecular and Cellular Biology, 13, 4531-4538), the MYC site (Agira et al., 1989, EMBO J., 8, 4273-4279), the OCT site (Rosales et al., 1987, EMBO J., 6, 3015-3025), the NF-GMa site (Shannon et al., 1988, Proc. Natl. Acad. Sci. USA, 85, 674-678), the GAS box (Pelligrini and Schindler, 1993, Trends Biochem. Sci., 18, 338-342), the E box (Blackwell et al., 1990, Science, 250, 1149-1151) and the NfκB site (Sen and Baltimore, 1986, Cell, 46, 705-716).

The present invention also relates to a nucleotide sequence which can include unnatural nucleotides, in particular sulphur-containing nucleotides, for example, or nucleotides having an α or β structure, or nucleotides which are labelled with a label which is, by way of example, selected from the group consisting of an enzyme, biotin, iminobiotin, a fluorescent compound, a radioactive compound, a chemiluminescent compound, an electrodense compound, a magnetic compound, an antigen, a hapten and an antibody.

The present invention also relates to cloning or expression vectors which include at least one nucleotide sequence such as previously described.

These cloning or expression vectors can additionally include elements which ensure expression of the sequence in the host cell, in particular promoter sequences and/or regulatory sequences which are effective in the said cell, if the sequence is a coding sequence.

›If the sequence is a sequence which possesses…

If the sequence is a sequence which possesses a transcriptional promoter activity, the vector will additionally include homologous or heterologous nucleic acid sequences which it is desired to express in the said cell.

Preferably, these cloning or expression vectors comprise at least one gene of interest which is placed under the control of at least one nucleic acid sequence which is as previously described and which exhibits a transcriptional promoter activity.

The said gene of interest can, for example, be selected from the group which consists of the genes which encode the CIITA factor and the α and β chains of the HLA-DR, HLA-DQ and/or HLA-DP molecules, and reporter genes, such as the gene which encodes rabbit β globin.

The vector in question can be selected from the autonomously replicating vectors or from the vectors which integrate into the chromosome.

In the case of an autonomously replicating system, use will preferably be made, depending on whether the cell host is prokaryotic or eukaryotic, of systems of the plasmid type or of viral systems, with it being possible for the viral vectors to be, in particular, adenoviruses, poxviruses or herpesviruses. The skilled person is familiar with the techniques which can be used for each of these viruses.

When it is desired to integrate the sequence into the host cell chromosomes, it will be necessary to provide one or more sequences derived from the cell host at each end of the nucleic acid sequence to be integrated in order to ensure recombination. These are also processes which are widely described in the prior literature. Use can, in particular, be made of systems of the plasmid or viral type such as, for example, retroviruses or AAVs (adenoassociated viruses).

The invention also relates to prokaryotic or eukaryotic cells which have been transformed with a vector such as previously described, in particular in order to ensure expression of at least one of the forms of the CIITA factor which have been identified in accordance with the invention.

Cells which can be used for implementing the invention and which may indeed be mentioned are prokaryotic cells, yeast cells and animal cells, in particular cultures of mammalian cells.

Preferably, the host cell is selected from the group consisting of dendritic cells, B lymphocytes, T lymphocytes, macrophages, monocytes, thymus epithelium cells, muscle cells, fibroblasts, endothelial cells and cancer cells, in particular melanoma cells.

The cells which have thus been obtained can be used to prepare natural or mutated CIITA polypeptides and also fragments of these polypeptides.

These cells can also be used as model cells for the purpose of studying the mechanisms of regulating the transcriptional promoter function of the sequences which have been identified in accordance with the invention and of identifying specific inhibitors whose action could possibly be targeted in a given cell type. These cells can additionally be used as model cells for the purpose of studying the interactions between the different CIITA factors which have been isolated, or their variants, and the regions which direct transcription of the genes encoding the MHC class II molecules, and, especially, for the purpose of selecting the variants of the CIITA factors which are able to act as agonists or antagonists on the CIITA receptor. These types of cell model can be constructed using known techniques of genetic manipulation. Furthermore, the use of such cell models with a view to testing pharmaceutical compounds is well known to the skilled person.

The present invention also relates to organisms, such as animals, in particular mice, whose genome has been genetically modified in order to integrate at least one of the nucleic acid sequences according to the invention. In this case, again, these animals can be used as model animals in order to test the efficacy of particular pharmaceutical products.

The present invention also relates to a process for producing a CIITA polypeptide, in particular as defined in SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18 or SEQ ID No. 19, or an allelic variant of one of these polypeptides, which process comprises (i) culturing a host cell, which has been transformed with a vector which includes a nucleic acid sequence encoding a CIITA polypeptide as previously described, under culture conditions which are appropriate for producing the said polypeptide, and (ii) recovering the said polypeptide.

The said polypeptide can be recovered intracellularly or extracellularly in the culture medium when the vector has been designed for assuring secretion of the polypeptide by the expedient, for example, of using a leader sequence, with the polypeptide being in the form of a prepolypeptide. The constructs which enable polypeptides to be secreted are known, both for prokaryotic systems and eukaryotic systems.

The present invention also relates to a CIITA polypeptide which can be obtained by implementing the abovedescribed process.

The present invention additionally relates to CIITA polypeptides which correspond to the previously described nucleic acid sequences and which are in unnatural form, i.e. they are not used in their natural environment but are obtained by purification from natural sources or else obtained by genetic recombination.

More specifically, the invention relates to a polypeptide which is characterized in that it comprises at least one sequence which is selected from:

a) an amino acid sequence which consists of SEQ ID No. 16, or its allelic variants,

b) an amino acid sequence which consists of a part of the sequence SEQ ID No. 16, or its allelic variants.

Preferably, a peptide of this nature is characterized in that at least one part of its sequence is defined in the space between amino acid 1 and amino acid 94 of the sequence SEQ ID No. 16.

The invention also relates to the same polypeptides which are obtained by chemical synthesis and which can include unnatural amino acids. The invention also relates to the said polypeptides which are in a form which is totally or partially retro and/or inverso and which exhibit an activity which is equivalent to that observed in the case of the native CIITA factor, or one of its variants, according to the present invention, or at least an immunological activity which is identical to that of the parent CIITA factor.

›Furthermore, the polypeptides, and more specifically their variants…

Furthermore, the polypeptides, and more specifically their variants, such as previously described, can exhibit the same function of transactivating the expression of the genes encoding MHC class II molecules as a CIITA factor or, at least, the same ability to bind to the specific site for binding a CIITA factor during expression of the said genes.

The present invention additionally relates to an antibody which is directed against any one of the previously described polypeptides or against a polypeptide which contains at least one mutation affecting the function of the CIITA factor, as described below, and, more specifically, to a polyclonal or monoclonal antibody which is obtained by the immunological reaction of a human or animal organism with an immunogenic agent which comprises at least one of the said polypeptides.

The invention also relates to molecules which are able to inhibit either the function of the polypeptides which have been identified according to the invention in activating expression of the genes encoding the MHC class II molecules or the ability of these polypeptides to bind to the CIITA-binding site. These molecules can be polypeptides which contain at least one mutation which affects the function of the CIITA factor. A modified polypeptide of this nature, which consists, for example, of a structural analogue of the said polypeptide, can act as a lure. The molecules can also be antibodies, such as presented above, which are able, for example, to block either all or part of the CIITA factor which is able to react with its specific receptor, or a region of the CIITA factor which is able to interact with at least one other transactivating factor during expression of the genes encoding the MHC class II molecules.

The invention also relates to molecules which are able specifically to inhibit expression of the genes which encode MHC class II molecules in dendritic cells. These molecules consist, in particular, of all or part of a nucleic acid sequence which contains at least one mutation which affects the transcriptional promoter function of the said sequence, with the mutation(s) being located in an identified nucleic acid sequence SEQ ID No. 4, or its complementary sequence.

The invention also relates to molecules which are able specifically to inhibit the induction, by cytokines, of the expression of the genes which encode MHC class II molecules. These molecules consist, in particular, of all or part of a nucleic acid sequence which contains at least one mutation which affects the transcriptional promoter function of the said sequence, with the mutation(s) being located in the identified nucleic acid sequence SEQ ID No. 6, or its complementary sequence.

The present invention also relates to pharmaceutical compositions which comprise, as the active principle, at least one substance such as a nucleic acid sequence or an inhibitory molecule as previously defined. More specifically, the invention relates to a pharmaceutical composition for treating disorders in which it is desired to increase expression of the genes encoding MHC class II molecules, in particular in one cell type, more specifically in a dendritic cell. Furthermore, it is possible to observe this increase in the expression of the genes encoding MHC class II molecules following induction by a cytokine, more specifically by interferon γ or interleukin 4, in particular when the said pharmaceutical composition comprises at least one substance which consists of a nucleic acid sequence which can be activated by the said cytokine, as previously described. The invention additionally relates to a said pharmaceutical composition for treating disorders in which it is desired to reduce expression of the genes encoding MHC class II molecules, more specifically to a pharmaceutical composition which comprises, as the active principle, a) either a nucleic acid sequence according to the invention, the sequence of which is modified such that the promoter activity of the said sequence is affected, or which leads to the production of an inactive CIITA polypeptide, as previously described, or b) an inactive CIITA polypeptide.

The invention furthermore relates to a vaccine which can be used, in particular, for treating cancer or autoimmune diseases, characterized in that it comprises at least one of the pharmaceutical compositions presented above.

Finally, the present invention relates, more specifically, to methods for diagnosing a predisposition to a disorder which is linked to a disturbance in the expression of the genes encoding MHC class II molecules, characterized in that a biological sample is taken from a patient, and the presence of at least one mutation, within either sequences which exhibit a transcriptional promoter activity or sequences which encode one of the identified CIITA factors according to the present invention, is determined by analysing the said nucleic acid sequences and comparing with the wild-type sequences according to the invention, with the presence of at least one such mutation being indicative of a predisposition of the said patient to the said disorder.

A large number of disorders which are directly or indirectly linked to a disturbance in the expression of the genes encoding MHC class II molecules have been described in the literature. We may cite, by way of example, disorders such as insulin-dependent diabetes, multiple sclerosis, rheumatoid arthritis and lupus erythematosus, one of the elements of which disorders could be overexpression of the genes encoding MHC class II molecules; or, conversely, the BLS (bare lymphocytes syndrome) syndrome, which is associated with a severe immunodeficiency.

Sought-after mutations which may be mentioned, more specifically, are mutations which affect the promoter function of nucleic acid sequences, mutations which affect the cellular specificity of the said promoter function, or mutations which affect induction of the said promoter function by a cytokine.

The analysed nucleic acid sequence can equally well be a genomic DNA, a cDNA or an RNA.

›The diagnostic tools which are based on the…

The diagnostic tools which are based on the present invention can be used to make a positive and differential diagnosis in a subject taken in isolation or else to make a presymptomatic diagnosis in a subject at risk.

There are, of course, a very large number of methods for demonstrating a mutation in a gene as compared with the natural gene; they can be implemented by studying the genomic DNA, the cDNA, the RNA and/or the polypeptide. They can, essentially, be divided into two broad categories, with the first type of method being that in which the presence of a mutation is detected by comparing the mutated sequence with the corresponding natural, unmutated sequence, and the second type being that in which the presence of the mutation is detected indirectly. Advantageously, the mutation can be detected by demonstrating mismatches, which are due to the presence of the mutation, after analysing by means of hybridization which is carried out using at least one oligonucleotide probe which is specific for the sought-after mutation.

In each of the cases, preference will in general be given to the methods in which all or part of the sequence corresponding to all or part of the identified sequences SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 is amplified prior to demonstrating the mutation. These amplification methods are well known.

Furthermore, the mutated CIITA factors which are found in subjects who are exhibiting disorders in the expression of the genes encoding MHC type II molecules can exhibit an antigenicity which is different from that of the identified natural CIITA factors SEQ ID No. 16, SEQ ID No. 17 or SEQ ID No. 18. It is therefore possible to diagnose or prognosticate susceptibility to disorders which are linked to deregulation of the expression of the genes encoding MHC type II molecules by demonstrating the presence of the product of the mutated CIITA gene, for example using antibodies, in particular monoclonal antibodies, as previously described.

Other characteristics and advantages of the present invention will be apparent from reading the following examples, which are illustrated by FIGS. 1 to 9 . Nevertheless, the invention is not limited to the contents of the said examples.

FIGURE LEGENDS

FIG. 1 depicts the four 5′ ends of the CIITA mRNAs which were identified as described in Example 1. The coding regions are indicated by the wide boxes while the 5′ untranslated regions are indicated by the smaller boxes. The non-homologous regions are shown as clearly filled-in regions. The diagram shows the positions of the two primers, P1 and P2, which were used for the RACE-PCR amplification.

FIG. 2 shows the sequence, SEQ ID NO. 26 and the positions of the different sites for binding known transcription factors which were identified on the sequence, of the 5′-flanking region of the type I CIITA gene. The main transcription initiation site is also indicated by an arrow at +1.

FIG. 3 shows the sequence, SEQ ID No. 27 and the positions of the different sites for binding known transcription factors which were identified on the sequence, of the 5′-flanking region of the type III CIITA gene. The main transcription initiation site is also indicated by an arrow at +1.

FIG. 4 shows the sequence, and the positions of the different sites for binding known transcription factors which were identified on the sequence, of the 5′-flanking region of the type IV CIITA gene. The main transcription initiation site is also indicated by an arrow at +1.

FIG. 5 is a diagrammatic representation of the probes which were used in the RNAse protection tests during the analysis of the expression profiles of the different CIITA mRNAs. The different probes are shown with their sizes “before” and “after” digestion by RNAse. Each of the probes corresponds to a part of exon 1 and has 226 bases which are common to each of the mRNAs.

FIG. 6 is a diagrammatic representation of the differential expression of the four types of CIITA transcript. The quantity of each of the mRNA types is indicated as a percentage as compared with the total quantity of CIITA expression, as measured using the internal control and after PhosphoImager quantification of the fragments which were obtained following the RNAse protection analysis.

FIG. 7 is a diagrammatic representation of the same type as that in FIG. 6 except that expression of the CIITA transcripts is observed following induction with interferon α (+IFNγ).

FIG. 8 depicts the organization of the IV wild-type and mutants human promoter IV. Sequences and positions of the conserved cis-acting elements are indicated. Ponctual mutations introduced in GAS element, E box and IRF-1 are shown below the wild-type sequence SEQ ID No. 29 with names of mutant constructs.

FIG. 9 depicts the functional analysis of wild-type and mutagenized Gm, Em and Im promoter IV. Stimulation index of the gene reporter expression is expressed in %. Plotted results are means of three independent experiments with standard deviations.

The invention is also illustrated by the sequence identifiers SEQ ID No. 1 to SEQ ID No. 25, which sequence identifiers depict:

SEQ ID No. 1 to SEQ ID No. 3: the sequences of the three types of cDNA corresponding to the CIITA genes (sequences designated I, II and IV in FIG. 1 ), which were identified in accordance with the invention;

SEQ ID No. 4 to SEQ ID No. 6: the sequences which were identified as exhibiting a transcriptional promoter activity in the form I, form II and form IV CIITA genes and which were designated PI, PII and PIV, respectively;

SEQ ID No. 7 to SEQ ID No. 10: the sequences which correspond, respectively, to the different CIITA genes of forms I to IV, which genes lack the sequences which exhibit a transcriptional promoter activity;

SEQ ID No. 11: the sequence which corresponds to the coding part of the form I CIITA gene;

SEQ ID No. 12: the sequence which corresponds to the coding part of the form II CIITA gene;

SEQ ID No. 13: the sequence corresponding to the coding part of the form III CIITA gene;

›SEQ ID No. 14: the sequence corresponding to…

SEQ ID No. 14: the sequence corresponding to the coding part of the form IV CIITA gene, including a untranslated part;

SEQ ID No. 15: a fragment of the SEQ ID No. 14 sequence, corresponding to nucleotides 901 to 3390, counting from the first nucleotide of SEQ ID No. 13;

SEQ ID No. 16: the translation of SEQ ID No. 11 into amino acids, corresponding to a form I CIITA factor which possesses 101 additional amino acids at the N-terminal end, as compared with SEQ ID No. 17;

SEQ ID No. 17: the translation into amino acids of the coding part of the form I to form IV CIITA genes, starting from an ATG located 21 bases downstream of the 5′ end of the common exon 2 (FIG. 1 );

SEQ ID No. 18: the translation of the form III CIITA gene into amino acids, starting from a second ATG, and corresponding to a CIITA factor which possesses 24 additional amino acids at the N-terminal end;

SEQ ID No. 19: the translation of SEQ ID No. 15 into amino acids

SEQ ID No. 20 to 25: PCR primers

EXAMPLES

›Examples21

›Example 1

The cytoplasmic or total RNAs were extracted from various cell lines: Raji (Burkitt's lymphoma), Mann (human B lymphocyte), CEM (T lymphoblastoid line), THPA (monocyte), PP2 (fibroblast), Me67 (melanoma) after induction with interferon γ and HUVEC (human endothelial cell) using the technique described by Wilkinson (1988, Nucleic Acid Res. 16, 10933). The total RNA derived from the BC1 cell line (dendritic cells) was prepared using a Trizol-containing reagent (Gibco BRL). The RNAs derived from human spleen, thymus, tonsil and kidney were kindly provided by P. Sapino.

The 5′ ends of the RNAs which were obtained were analysed by the RACE PCR technique (Frohman et al., 1988, Proc. Natl. Acad. Sci, USA, 85, 8998-9002) in accordance with the manufacturer's (Gibco BRL) instructions, with the following modifications. After reverse transcription of the RNAs, and before the amplification step, a dATP tail is added to the ends of the cDNAs. During the PCR amplification, 5 μl of isolated cDNA-dA are added to 40 μl of an amplification mixture containing 200 μM of each of the dNTPs and 25 pmol of primers which are specific for the gene encoding the CIITA factor, i.e. P1 (5′-GGTCCAGTTCCGCGATATTGG-3′) SEQ ID NO. 20 and P2 (5′-TCCCTGGTCTCTTCATCA-3′), SEQ ID No. 21 25 pmol of adaptation primer ADXSC (5′-GACTCGAGTCGACATCG-3′) SEQ ID No. 22 and 10 pmol of adaptation primer XSCT17 (5′-GACTCGAGTCGACATCGAT-3′) SEQ ID NO. 23. After a preincubation at 95° C. for 5 minutes, 2 units of Taq polymerase are added and the amplification is carried out in 30 cycles of 45 seconds at 94° C., 25 seconds at 54° C. and 2 minutes at 72° C. The final incubation is carried out at 72° C. for 10 minutes.

These amplifications demonstrated the existence of four types of cDNA which corresponded to the CIITA factor. Analysis of the sequences of these nucleic acids showed that while these nucleic acids all possessed a common 3′ end (Exon 2), they diverged completely at their 5′ ends, thereby defining four different types of Exon 1. These four sequences (variable Exon 1+ common Exon 2) are identified as I, II, III and IV (FIG. 1 ).

As the analysis of the sequences indicates, these four transcripts, i.e., I, II, III and IV, exhibit a common reading frame which begins at the ATG which is located 21 bases downstream of the 5′ end of the common Exon 2 (FIG. 1 ). In the case of sequences II and IV, this ATG is the first initiation codon. In the case of sequences I and III, another ATG exists which leads to the synthesis of a CIITA factor which possesses 101 or 24 additional amino acids, respectively, at the N-terminal end of the translated polypeptide.

›Example 2

The sites for initiating transcription of the different human CIITA mRNAs which had been identified were tested by means of RNAse protection using DNA fragments which were specific for the different Exons 1.

In the case of the type I transcripts, three protected fragments were identified using nucleic acid which was isolated from liver. The major fragment corresponds to the transcription initiation site which is located 380 bases upstream of the 3′ end of Exon 1 (FIG. 1 ). This site was defined as nucleotide +1 of the type I mRNAs. The two other transcripts are obtained from initiation sites which were located in positions −14 and +8. The locations of these initiation sites are compatible with the use, during translation, of the ATG signals identified in Example 1.

In the case of the type III transcripts, several protected fragments were identified using nucleic acid which was isolated from B lymphocytes. The major transcript corresponds to an initiation which starts from the position 183 bases upstream of the 3′ end of Exon 1 and defines position +1 of the type III transcripts. Two other initiation sites are located at positions −8 and −4. Other minor sites are identified in positions −23 and +34. These initiation sites are compatible with the use of the two ATG sites located in Example 1.

In the case of the type IV transcripts, a large number of protected fragments were identified using nucleic acids which were isolated from melanoma cells which were induced with interferon γ. The major transcript corresponds to a transcription initiation which is located 75 bases upstream of the 3′ end of Exon 1, which site defines position +1 of the type IV transcripts. A second major initiation site is observed at position +17, as well as six minor sites which are located between positions −54 and +69 of Exon 1. These initiation sites are compatible with the use of the ATG which is located 21 bases downstream of the 5′ end of Exon 2 (Example 1).

The presence of distinct initiation sites for each of the I, II, III and IV RNAs suggests that the promoter regions which control expression of the corresponding genes are distinct (designated P1, PII, PIII and PIV).

›Example 3

Having identified the sequence divergences which were observed at the 5′ ends of the mRNAs (Exon 1 and untranslated sequence), the applicant then isolated the genomic sequences, including the promoter regions, of genes I, II, III and IV from a λ phage library containing the human genome.

Comparison of the sequences corresponding to the four promoters PI, PII, PIII and PIV does not demonstrate any significant homology. None of these regions contains a GC or TATA box. This latter observation explains the substantial number of initiation sites which were observed for a given transcript.

By contrast, it was possible to identify several sites corresponding to sites for binding elements which act in cis during the transcription of other genes. Thus, promoter PI contains an NF-GMb site, an NF-IL6 site, two inverted NF-IL6 sites, a PEA3 site, and a PEA3 site in the opposite direction, an AP1 site and a CCAAT box (FIG. 2 ). Similarly, promoter PIII contains an E2A box in the opposite direction, an IRF1/2 site, an MYC site in the opposite direction and an OCT site in the opposite direction (FIG. 3 ). An NF-GMa site, a GAS box, an E box, a IRF1/2 site and an NfKB site are found in promoter IV (FIG. 4 ).

›Example 4

In order to study the expression profile of these different genes in various cell types, four cDNA fragments which were specific for each of the mRNA forms were prepared as RNAse protection probes. These probes are depicted in FIG. 5 . Use is made of an internal control which makes it possible to evaluate the total expression of the CIITA-encoding genes (from nucleotide 1152, PstI site, to nucleotide 1344, NcoI site, protecting 193 bases of the region possessed in common by the RNAs (Exon 2)). The RNAse protection tests are carried out on 25 μg of RNA as previously described (Steimle et al. , 1993, Cell, 75, 135-146). The results are quantified by using a PhosphorImager. The promoter function is quantified as being the ratio of the expression of a specific type of mRNA as compared with the total expression of the CIITA-encoding genes measured using the internal control.

An analysis was carried out of the mRNAs which were derived from different tissues or cell lines which were expressing the CIITA gene either constitutively or following induction with interferon γ.

The results (Table 1 and FIG. 6) show that differential use of the PI, PII, PIII and PIV promoters occurs. Thus, it was shown that the type I mRNAs, which result from using PI, are very strongly expressed in dendritic cells (FIG. 6 ), more weakly expressed in the spleen and thymus, and not expressed at all in the other tissues or cell lines.

The type III mRNAs are detected at a high level in different B lymphocyte cell lines as well as in tissues which are rich in B lymphocytes, such as the spleen and the tonsils, or the thymus (FIG. 6 ). By contrast, these type III mRNAs are very weakly expressed in dendritic cells or in cells which can be induced with interferon γ (Me67.1, THP1, HUVEC and PP2).

The type IV mRNAs are the form which is principally expressed following induction with interferon A. That this was the case was observed in a variety of inducible cell lines such as Me67.1 (melanoma), THP1 (monocyte), HUVEC (endothelial cells) and PP2 (fibroblasts). By contrast, these mRNAs are only weakly expressed in B lymphocytes or dendritic cells (FIG. 5 ).

›Example 5

The functional activity and the tissue specificity of the PIII and PIV promoters were analysed by transfecting cells with constructs which combined a reporter gene and a promoter. Given the fact that the type III mRNAs are mainly expressed in B lymphocytes and that the type IV mRNAs are preferentially expressed in cells which can be induced with interferon γ, the test cell lines selected are the Raji (B lymphocyte) and Me67.8 (melanoma) cell lines. The reporter gene which is selected is the gene which encodes rabbit β globin. The promoter region to be tested is cloned upstream of this gene into plasmid PGβG(+) (Sperisen et al., 1992, PCR. Methods Appl. 1, 164-170). The plasmids pIII-974 and pIII-322 contain the −974 (NheI)/+101 (HpaII) and −322 (PstI)/+101 (HpaII) fragments, respectively, of the genome regions which are located 5′ of the type III Exon 1. Plasmids pIV-950 and pIV-461 contain the −950 (XhoI)/+75 and −461 (KpnI)/+75 fragments, respectively, of the genome regions located 5′ of the type IV Exon 1. A reference plasmid is also used as a control: this is a plasmid which contains a gene encoding rabbit β globin which possesses a deletion of 40 bases and which is transcribed under the control of a constitutive chicken promoter (pGβAcβGID, Sperisen et al., 1992). Expression of the reporter gene is measured by quantitative RT-PCR as described in Sperisen et al., 1992 with the following modifications. 5×10 6 Raji cells and 2.5×10 6 Me67.8 cells were transfected, by electroporation at 250V and 960 μF (GenePulse, BioRad), with 20 μg of a plasmid preparation, which consisted of a defined ratio of the plasmid as previously described and the reference plasmid, and 400 μg of E. Coli tRNA, as the carrier molecule, in 750 μl of RPMI buffer. For the step of induction with interferon γ, the cell cultures are placed, following transformation, in the presence (500U/ml) or absence of the inducer. The cells are cultured at 37° C. for 48 hours. The total RNAs are extracted with the Triazol-containing reagent and digested with RNAse-free DNAseI (Boehringer). 1 μg of total RNA is used for carrying out the reverse transcription in the presence of a primer (dT) 20 and RNAse-free Superscript (50U, GIBCO BRL) reverse transcriptase and 10U of RNAse inhibitor. Subsequently, 1/10 of the cDNA which has been obtained is amplified using the primers βGP5′ (5′-TCCCCCAAAACAGACAGAATGG-3′) SEQ ID No. 24 (40 pmol) and βGP3′ (5′-GTCACAGTGCAGTTCACTCAG-3′) SEQ ID No. 25 (40 pmol) in a 50 μl volume containing 5 μl of 10×Vent buffer in the presence of 2 μCi of (α 32 P)dCTP (Amersham). After preincubating at 95° C. for 3 minutes, 2U of Vent DNA polymerase (NEB) are added. The amplification is carried out in 30 cycles of 40 seconds at 94° C., 30 seconds at 59° C. and 60 seconds at 72° C. The PCR products are denatured and loaded onto a denaturing polyacrylamide gel (6%, 8M urea). The signals are quantified using the PhosphoImager.

The results obtained show that while transfection of B lymphocytes with plasmid pIII-974 and pIII-322 is accompanied by strong activity of the pIII promoter, the same promoters are inactive in Me67.8 cells before or after induction. It is furthermore observed that pIII-322 is expressed better than plasmid pIII-974 in the B lymphocytes.

By contrast, when plasmids pIV-950 and pIV-461 are used, only basal expression is observed in the B lymphocytes whereas very strong expression is observed in the induced Me67.8 cells and in other types of induced cells (Hela or 2FTGH). Moreover, the expression signals of these two plasmids pIV-950 and pIV-461 have values of 0.13 and 0.18, respectively, before induction and of 7.9 and 29.6, respectively, following induction with interferon.

›Example 6

As shown in FIG. 4, SEQ ID No. 6, corresponding to cytokine inducible CIITA promoter IV, contains at least 3 potential cis-acting elements which could be involved in transcription regulation of a gene located downstream of the said sequence. These elements are GAS element, E box and IRF-1 binding site.

In order to analyse the functional relevance of these cis-acting elements, site directed mutagenesis was performed. A reporter gene has been constructed. This plasmid contains the −308 to +75 fragment of SEQ ID No. 6 subcloned upstream of the rabbit β-globin gene of plasmid pGβG(+). Directed mutagenesis was performed introducing many ponctual mutations in sequences to be analysed in the said plasmid (see FIG. 8) leading to 3 mutants named Gm, Em and Im corresponding to mutations in GAS element, E box and IRF-1, respectively.

Reporter gene (wild type and each of the mutated plasmids) expression was measured after gamma interferon activation, in cell line Me67.8 (melanoma cell line) by quantitative RT-PCR (Sperisen et al., 1992, PCR. Meth. Appli., 1, 164-170). Transfections, induction, RNA preparation and RT-PCR analyses were performed as previously described (Muhlenthaler-Motter et al., 1997, EMBO J., 16, 2851-2860).

Transfections of the wild-type plasmid in the melanoma cell line Me67.8, without gamma interferon activation, showed a very low basal transcriptional activity of the β-globin reporter gene (not shown). Treatment of these transfected cells with gamma interferon induced a very high expression of the β-globulin gene showing a strong activity of promoter IV (the activity was referred as 100% of stimulation index).

Similarly, β-globin reporter gene expression directed by mutated sequences (Gm, Em and Im) was analysed. Results (FIG. 9) showed that mutations in either the GAS element (Gm), the E-box (Em) or IRF-1 (Im) of SEQ ID No. 6 resulted in an almost complete abolition of promoter inducibility by gamma interferon, with stimulation index equivalent to 19%, 16% and 23% of wild-type stimulation index, respectively. The same reduction of promoter responsiveness to gamma interferon was observed with a double mutant GmEm (GAS element and E-box) leading to 17% of the wild-type stimulation index.

These results show that each of these cis-acting elements are functionally relevant for gamma interferon stimulation of expression of gene located downstream of promoter IV.

›Example 7 · 1 of 15

Since we have demonstrated the functional importance of the IRF-1 binding site of promoter IV and since IRF-1 was shown to be involved in the induction of several interferon gamma inducible genes, such as for example GBP (Briken et al., 1995, Mol. Cell. Biol., 15, 975-982), we investigated the role of IRF-1 in the induction of CIITA by interferon gamma. RNAs from embryonic fibroblasts (EF) derived from wild-type (wt) and from IRF-1 − mice (which do not express IRF-1) were compared for CIITA mRNA expression stimulation by interferon gamma. RNAse protection assays revealed that, in contrast to wild-type EF, interferon gamma induced CIITA mRNA expression was strongly reduced in IRF-1 − EF. The same inhibition of interferon gamma stimulation was observed for GBP mRNA. The results indicate that IRF-1 is an essential factor for induction efficiency by interferon gamma.

5463 base pairs

nucleic acid

single

linear

DNA (genomic)

cIIta gene of type I

GTAAGTACTG TAACAGAGAC TAAATGCTAA GTAAGGCAGG CGTGGTGGCT CACACTTGTA 60

ATCCCAGTAC TTTGGAGGAC TGAGGCAAGA GGATCACTTG AGCCCAGAAT TCAAGACCAG 120

CCTGGGAAAC AGAGTGAGAC CCTGTCTCTA CTAAAAATAA AAAAATAAAA AATTAGCGGG 180

GCATGATGGT GTGAGTCTGT AGTCCCAGCT ACTGGGAAAC AGAGATGGGA GGTTTGTTTG 240

AGCCTGGGAA GTTGAGGCTG CAGTGAGTTA TGATCACGCC ACTGCACACC AGCCTGGGCA 300

ACAGAGCAAG ACCCTGTCAA AAAAAAAAAA AAGGCTATGT AACACCCCCA AGTCACATAA 360

TTGGCAAGGA GCAGCAGATC TGGGACTTGA ACATAGGCAG ATTAGCTCCA AGCCTATTTG 420

CTTAACCTCT ATACCACAAT GCCTTCTTGC TATGGTAAAA CATCTGAAAA GACCTATTAC 480

CCTATAGGTC CTCTAAGGAG GCATGTCGCC TTCCTCTTAG CAATACTAGA TTGGCTCCAA 540

CAGAAGGCTG TGGGCTTCTC TGGCACATGC ACCTGGGTAG GACCCAGAGA ATATCTGTGG 600

AGTCTGAATC AACCCAAAAG CCAATATCCA TCCGTTCATC AGGAACCCCA GCCTACAACG 660

CAAAAGAGGA AATCTTCCTA AGTAGAAATA AACTGTAATA AATTGCAGAG GTTCCCTCGT 720

CCTGGTTTTC ACTTCATGTT TTGGATGCTG CATGCTGGGT GAGCGGAGAT TCCAGGCACT 780

GGCCAGGGCA GCTGCCCTGA CTCCAAGGGC TGCCATGAAC AACTTCCAGG CCATCCTGAC 840

TCAGGTGAGA ATGCTGCTCT CCAGCCATCA GCCCAGCCTG GTGCAGGCCC TCTTGGACAA 900

CCTGCTGAAG GAGGACCTCC TCTCCAGGGA ATACCACTGC ACTCTGCTCC ATGAGCCTGA 960

TAGTGAGGCT CTGGCCAGGA AGATCTCTTT GACCCTACTA GAGAAAGGAG ACCTGGATTT 1020

GGCCCTCCTG GGGTGGGCCC GGAGTGGGCT GCAGCCCCCA GCAGCCGAGA GGGGCCCCGG 1080

CCACAGTGAC CATGGTGGCA GCTCACAGTG TGCCACCATG GAGTTGGGGC CCCTAGAAGG 1140

TGGCTACCTG GAGCTTCTTA ACAGCGATGC TGACCCCCTG TGCCTCTACC ACTTCTATGA 1200

CCAGATGGAC CTGGCTGGAG AAGAAGAGAT TGAGCTCTAC TCAGAACCCG ACACAGACAC 1260

CATCAACTGC GACCAGTTCA GCAGGCTGTT GTGTGACATG GAAGGTGATG AAGAGACCAG 1320

GGAGGCTTAT GCCAATATCG CGGAACTGGA CCAGTATGTC TTCCAGGACT CCCAGCTGGA 1380

GGGCCTGAGC AAGGACATTT TCAAGCACAT AGGACCAGAT GAAGTGATCG GTGAGAGTAT 1440

GGAGATGCCA GCAGAAGTTG GGCAGAAAAG TCAGAAAAGA CCCTTCCCAG AGGAGCTTCC 1500

GGCAGACCTG AAGCACTGGA AGCCAGCTGA GCCCCCCACT GTGGTGACTG GCAGTCTCCT 1560

AGTGGGACCA GTGAGCGACT GCTCCACCCT GCCCTGCCTG CCACTGCCTG CGCTGTTCAA 1620

CCAGGAGCCA GCCTCCGGCC AGATGCGCCT GGAGAAAACC GACCAGATTC CCATGCCTTT 1680

CTCCAGTTCC TCGTTGAGCT GCCTGAATCT CCCTGAGGGA CCCATCCAGT TTGTCCCCAC 1740

CATCTCCACT CTGCCCCATG GGCTCTGGCA AATCTCTGAG GCTGGAACAG GGGTCTCCAG 1800

TATATTCATC TACCATGGTG AGGTGCCCCA GGCCAGCCAA GTACCCCCTC CCAGTGGATT 1860

CACTGTCCAC GGCCTCCCAA CATCTCCAGA CCGGCCAGGC TCCACCAGCC CCTTCGCTCC 1920

ATCAGCCACT GACCTGCCCA GCATGCCTGA ACCTGCCCTG ACCTCCCGAG CAAACATGAC 1980

AGAGCACAAG ACGTCCCCCA CCCAATGCCC GGCAGCTGGA GAGGTCTCCA ACAAGCTTCC 2040

AAAATGGCCT GAGCCGGTGG AGCAGTTCTA CCGCTCACTG CAGGACACGT ATGGTGCCGA 2100

GCCCGCAGGC CCGGATGGCA TCCTAGTGGA GGTGGATCTG GTGCAGGCCA GGCTGGAGAG 2160

GAGCAGCAGC AAGAGCCTGG AGCGGGAACT GGCCACCCCG GACTGGGCAG AACGGCAGCT 2220

GGCCCAAGGA GGCCTGGCTG AGGTGCTGTT GGCTGCCAAG GAGCACCGGC GGCCGCGTGA 2280

GACACGAGTG ATTGCTGTGC TGGGCAAAGC TGGTCAGGGC AAGAGCTATT GGGCTGGGGC 2340

AGTGAGCCGG GCCTGGGCTT GTGGCCGGCT TCCCCAGTAC GACTTTGTCT TCTCTGTCCC 2400

CTGCCATTGC TTGAACCGTC CGGGGGATGC CTATGGCCTG CAGGATCTGC TCTTCTCCCT 2460

GGGCCCACAG CCACTCGTGG CGGCCGATGA GGTTTTCAGC CACATCTTGA AGAGACCTGA 2520

CCGCGTTCTG CTCATCCTAG ACGCCTTCGA GGAGCTGGAA GCGCAAGATG GCTTCCTGCA 2580

CAGCACGTGC GGACCGGCAC CGGCGGAGCC CTGCTCCCTC CGGGGGCTGC TGGCCGGCCT 2640

TTTCCAGAAG AAGCTGCTCC GAGGTTGCAC CCTCCTCCTC ACAGCCCGGC CCCGGGGCCG 2700

CCTGGTCCAG AGCCTGAGCA AGGCCGACGC CCTATTTGAG CTGTCCGGCT TCTCCATGGA 2760

GCAGGCCCAG GCATACGTGA TGCGCTACTT TGAGAGCTCA GGGATGACAG AGCACCAAGA 2820

CAGAGCCCTG ACGCTCCTCC GGGACCGGCC ACTTCTTCTC AGTCACAGCC ACAGCCCTAC 2880

TTTGTGCCGG GCAGTGTGCC AGCTCTCAGA GGCCCTGCTG GAGCTTGGGG AGGACGCCAA 2940

GCTGCCCTCC ACGCTCACGG GACTCTATGT CGGCCTGCTG GGCCGTGCAG CCCTCGACAG 3000

CCCCCCCGGG GCCCTGGCAG AGCTGGCCAA GCTGGCCTGG GAGCTGGGCC GCAGACATCA 3060

AAGTACCCTA CAGGAGGACC AGTTCCCATC CGCAGACGTG AGGACCTGGG CGATGGCCAA 3120

AGGCTTAGTC CAACACCCAC CGCGGGCCGC AGAGTCCGAG CTGGCCTTCC CCAGCTTCCT 3180

CCTGCAATGC TTCCTGGGGG CCCTGTGGCT GGCTCTGAGT GGCGAAATCA AGGACAAGGA 3240

GCTCCCGCAG TACCTAGCAT TGACCCCAAG GAAGAAGAGG CCCTATGACA ACTGGCTGGA 3300

GGGCGTGCCA CGCTTTCTGG CTGGGCTGAT CTTCCAGCCT CCCGCCCGCT GCCTGGGAGC 3360

CCTACTCGGG CCATCGGCGG CTGCCTCGGT GGACAGGAAG CAGAAGGTGC TTGCGAGGTA 3420

CCTGAAGCGG CTGCAGCCGG GGACACTGCG GGCGCGGCAG CTGCTTGAGC TGCTGCACTG 3480

CGCCCACGAG GCCGAGGAGG CTGGAATTTG GCAGCACGTG GTACAGGAGC TCCCCGGCCG 3540

CCTCTCTTTT CTGGGCACCC GCCTCACGCC TCCTGATGCA CATGTACTGG GCAAGGCCTT 3600

GGAGGCGGCG GGCCAAGACT TCTCCCTGGA CCTCCGCAGC ACTGGCATTT GCCCCTCTGG 3660

ATTGGGGAGC CTCGTGGGAC TCAGCTGTGT CACCCGTTTC AGGGCTGCCT TGAGCGACAC 3720

GGTGGCGCTG TGGGAGTCCC TGCGGCAGCA TGGGGAGACC AAGCTACTTC AGGCAGCAGA 3780

GGAGAAGTTC ACCATCGAGC CTTTCAAAGC CAAGTCCCTG AAGGATGTGG AAGACCTGGG 3840

AAAGCTTGTG CAGACTCAGA GGACGAGAAG TTCCTCGGAA GACACAGCTG GGGAGCTCCC 3900

TGCTGTTCGG GACCTAAAGA AACTGGAGTT TGCGCTGGGC CCTGTCTCAG GCCCCCAGGC 3960

TTTCCCCAAA CTGGTGCGGA TCCTCACGGC CTTTTCCTCC CTGCAGCATC TGGACCTGGA 4020

TGCGCTGAGT GAGAACAAGA TCGGGGACGA GGGTGTCTCG CAGCTCTCAG CCACCTTCCC 4080

CCAGCTGAAG TCCTTGGAAA CCCTCAATCT GTCCCAGAAC AACATCACTG ACCTGGGTGC 4140

CTACAAACTC GCCGAGGCCC TGCCTTCGCT CGCTGCATCC CTGCTCAGGC TAAGCTTGTA 4200

CAATAACTGC ATCTGCGACG TGGGAGCCGA GAGCTTGGCT CGTGTGCTTC CGGACATGGT 4260

GTCCCTCCGG GTGATGGACG CAAGTTCACG GCTGCCGGGG CCCAGCAGCT CGCTGCCAGC 4320

CTTCGGAGGT GTCCTCATGT GGAGACGCTG GCGATGTGGA CGCCCACCAT CCCATTCAGT 4380

›Example 7 · 2 of 15

GTCCAGGAAC ACCTGCAACA ACAGGATTCA CGGATCAGCC TGAGATGATC CCAGCTGTGC 4440

TCTGGACAGG CATGTTCTCT GAGGACACTA ACCACGCTGG ACCTTGAACT GGGTACTTGT 4500

GGACACAGCT CTTCTCCAGG CTGTATCCCA TGAGGCCTCA GCATCCTGGC ACCCGGCCCC 4560

TGCTGGTTCA GGGTTGGCCC CTGCCCGGCT GCGGAATGAA CCACATCTTG CTCTGCTGAC 4620

AGACACAGGC CCGGCTCCAG GCTCCTTTAG CGCCCAGTTG GGTGGATGCC TGGTGGCAGC 4680

TGCGGTCCAC CCAGGAGCCC CGAGGCCTTC TCTGAAGGAC ATTGCGGACA GCCACGGCCA 4740

GGCCAGAGGG AGTGACAGAG GCAGCCCCAT TCTGCCTGCC CAGGCCCCTG CCACCCTGGG 4800

GAGAAAGTAC TTCTTTTTTT TTATTTTTAG ACAGAGTCTC ACTGTTGCCC AGGCTGGCGT 4860

GCAGTGGTGC GATCTGGGTT CACTGCAACC TCCGCCTCTT GGGTTCAAGC GATTCTTCTG 4920

CTTCAGCCTC CCGAGTAGCT GGGACTACAG GCACCCACCA TCATGTCTGG CTAATTTTTC 4980

ATTTTTAGTA GAGACAGGGT TTTGCCATGT TGGCCAGGCT GGTCTCAAAC TCTTGACCTC 5040

AGGTGATCCA CCCACCTCAG CCTCCCAAAG TGCTGGGGAT TACAAGCGTG AGCCACTGCA 5100

CCGGGCCACA GAGAAAGTAC TTCTCCACCC TGCTCTCCGA CCAGACACCT TGACAGGGCA 5160

CACCGGGCAC TCAGAAGACA CTGATGGGCA ACCCCCAGCC TGCTAATTCC CCAGATTGCA 5220

ACAGGCTGGG CTTCAGTGGC AGGCTGCTTT TGTCTATGGG ACTCAATGCA CTGACATTGT 5280

TGGCCAAAGC CAAAGCTAGG CCTGGCCAGA TGCACCAGGC CCTTAGCAGG GAAACAGCTA 5340

ATGGGACACT AATGGGGCGG TGAGAGGGGA ACAGACTGGA AGCACAGCTT CATTTCCTGT 5400

GTCTTTTTTC ACTACATTAT AAATGTCTCT TTAATGTCAC AAAAAAAAAA AAAAAAAAAA 5460

AAA 5463

4564 base pairs

nucleic acid

single

linear

DNA (genomic)

cIIta gene of type II

CCCGGGCGCC CCGCCTCAGT TTCCCCATCT ATAAAGTGGA GATGATAATA GCATTCAGAG 60

TCACTGATCT AAGGGCTCAG GGACACCATT CAGTGTAAGC CCCATACACT CCCTGCAAGA 120

GGAAGCTGGT TCTGACTCAG CCTTGAGGCT GGCGTCTGAG GCAACCACAA GCCCAACGTG 180

CATGGTGGAA AGATGACTGC AGCTCACAGT GTGCCACCAT GGAGTTGGGG CCCCTAGAAG 240

GTGGCTACCT GGAGCTTCTT AACAGCGATG CTGACCCCCT GTGCCTCTAC CACTTCTATG 300

ACCAGATGGA CCTGGCTGGA GAAGAAGAGA TTGAGCTCTA CTCAGAACCC GACACAGACA 360

CCATCAACTG CGACCAGTTC AGCAGGCTGT TGTGTGACAT GGAAGGTGAT GAAGAGACCA 420

GGGAGGCTTA TGCCAATATC GCGGAACTGG ACCAGTATGT CTTCCAGGAC TCCCAGCTGG 480

AGGGCCTGAG CAAGGACATT TTCAAGCACA TAGGACCAGA TGAAGTGATC GGTGAGAGTA 540

TGGAGATGCC AGCAGAAGTT GGGCAGAAAA GTCAGAAAAG ACCCTTCCCA GAGGAGCTTC 600

CGGCAGACCT GAAGCACTGG AAGCCAGCTG AGCCCCCCAC TGTGGTGACT GGCAGTCTCC 660

TAGTGGGACC AGTGAGCGAC TGCTCCACCC TGCCCTGCCT GCCACTGCCT GCGCTGTTCA 720

ACCAGGAGCC AGCCTCCGGC CAGATGCGCC TGGAGAAAAC CGACCAGATT CCCATGCCTT 780

TCTCCAGTTC CTCGTTGAGC TGCCTGAATC TCCCTGAGGG ACCCATCCAG TTTGTCCCCA 840

CCATCTCCAC TCTGCCCCAT GGGCTCTGGC AAATCTCTGA GGCTGGAACA GGGGTCTCCA 900

GTATATTCAT CTACCATGGT GAGGTGCCCC AGGCCAGCCA AGTACCCCCT CCCAGTGGAT 960

TCACTGTCCA CGGCCTCCCA ACATCTCCAG ACCGGCCAGG CTCCACCAGC CCCTTCGCTC 1020

CATCAGCCAC TGACCTGCCC AGCATGCCTG AACCTGCCCT GACCTCCCGA GCAAACATGA 1080

CAGAGCACAA GACGTCCCCC ACCCAATGCC CGGCAGCTGG AGAGGTCTCC AACAAGCTTC 1140

CAAAATGGCC TGAGCCGGTG GAGCAGTTCT ACCGCTCACT GCAGGACACG TATGGTGCCG 1200

AGCCCGCAGG CCCGGATGGC ATCCTAGTGG AGGTGGATCT GGTGCAGGCC AGGCTGGAGA 1260

GGAGCAGCAG CAAGAGCCTG GAGCGGGAAC TGGCCACCCC GGACTGGGCA GAACGGCAGC 1320

TGGCCCAAGG AGGCCTGGCT GAGGTGCTGT TGGCTGCCAA GGAGCACCGG CGGCCGCGTG 1380

AGACACGAGT GATTGCTGTG CTGGGCAAAG CTGGTCAGGG CAAGAGCTAT TGGGCTGGGG 1440

CAGTGAGCCG GGCCTGGGCT TGTGGCCGGC TTCCCCAGTA CGACTTTGTC TTCTCTGTCC 1500

CCTGCCATTG CTTGAACCGT CCGGGGGATG CCTATGGCCT GCAGGATCTG CTCTTCTCCC 1560

TGGGCCCACA GCCACTCGTG GCGGCCGATG AGGTTTTCAG CCACATCTTG AAGAGACCTG 1620

ACCGCGTTCT GCTCATCCTA GACGCCTTCG AGGAGCTGGA AGCGCAAGAT GGCTTCCTGC 1680

ACAGCACGTG CGGACCGGCA CCGGCGGAGC CCTGCTCCCT CCGGGGGCTG CTGGCCGGCC 1740

TTTTCCAGAA GAAGCTGCTC CGAGGTTGCA CCCTCCTCCT CACAGCCCGG CCCCGGGGCC 1800

GCCTGGTCCA GAGCCTGAGC AAGGCCGACG CCCTATTTGA GCTGTCCGGC TTCTCCATGG 1860

AGCAGGCCCA GGCATACGTG ATGCGCTACT TTGAGAGCTC AGGGATGACA GAGCACCAAG 1920

ACAGAGCCCT GACGCTCCTC CGGGACCGGC CACTTCTTCT CAGTCACAGC CACAGCCCTA 1980

CTTTGTGCCG GGCAGTGTGC CAGCTCTCAG AGGCCCTGCT GGAGCTTGGG GAGGACGCCA 2040

AGCTGCCCTC CACGCTCACG GGACTCTATG TCGGCCTGCT GGGCCGTGCA GCCCTCGACA 2100

GCCCCCCCGG GGCCCTGGCA GAGCTGGCCA AGCTGGCCTG GGAGCTGGGC CGCAGACATC 2160

AAAGTACCCT ACAGGAGGAC CAGTTCCCAT CCGCAGACGT GAGGACCTGG GCGATGGCCA 2220

AAGGCTTAGT CCAACACCCA CCGCGGGCCG CAGAGTCCGA GCTGGCCTTC CCCAGCTTCC 2280

TCCTGCAATG CTTCCTGGGG GCCCTGTGGC TGGCTCTGAG TGGCGAAATC AAGGACAAGG 2340

AGCTCCCGCA GTACCTAGCA TTGACCCCAA GGAAGAAGAG GCCCTATGAC AACTGGCTGG 2400

AGGGCGTGCC ACGCTTTCTG GCTGGGCTGA TCTTCCAGCC TCCCGCCCGC TGCCTGGGAG 2460

CCCTACTCGG GCCATCGGCG GCTGCCTCGG TGGACAGGAA GCAGAAGGTG CTTGCGAGGT 2520

ACCTGAAGCG GCTGCAGCCG GGGACACTGC GGGCGCGGCA GCTGCTTGAG CTGCTGCACT 2580

GCGCCCACGA GGCCGAGGAG GCTGGAATTT GGCAGCACGT GGTACAGGAG CTCCCCGGCC 2640

GCCTCTCTTT TCTGGGCACC CGCCTCACGC CTCCTGATGC ACATGTACTG GGCAAGGCCT 2700

TGGAGGCGGC GGGCCAAGAC TTCTCCCTGG ACCTCCGCAG CACTGGCATT TGCCCCTCTG 2760

GATTGGGGAG CCTCGTGGGA CTCAGCTGTG TCACCCGTTT CAGGGCTGCC TTGAGCGACA 2820

CGGTGGCGCT GTGGGAGTCC CTGCGGCAGC ATGGGGAGAC CAAGCTACTT CAGGCAGCAG 2880

AGGAGAAGTT CACCATCGAG CCTTTCAAAG CCAAGTCCCT GAAGGATGTG GAAGACCTGG 2940

GAAAGCTTGT GCAGACTCAG AGGACGAGAA GTTCCTCGGA AGACACAGCT GGGGAGCTCC 3000

CTGCTGTTCG GGACCTAAAG AAACTGGAGT TTGCGCTGGG CCCTGTCTCA GGCCCCCAGG 3060

CTTTCCCCAA ACTGGTGCGG ATCCTCACGG CCTTTTCCTC CCTGCAGCAT CTGGACCTGG 3120

ATGCGCTGAG TGAGAACAAG ATCGGGGACG AGGGTGTCTC GCAGCTCTCA GCCACCTTCC 3180

CCCAGCTGAA GTCCTTGGAA ACCCTCAATC TGTCCCAGAA CAACATCACT GACCTGGGTG 3240

CCTACAAACT CGCCGAGGCC CTGCCTTCGC TCGCTGCATC CCTGCTCAGG CTAAGCTTGT 3300

ACAATAACTG CATCTGCGAC GTGGGAGCCG AGAGCTTGGC TCGTGTGCTT CCGGACATGG 3360

TGTCCCTCCG GGTGATGGAC GCAAGTTCAC GGCTGCCGGG GCCCAGCAGC TCGCTGCCAG 3420

CCTTCGGAGG TGTCCTCATG TGGAGACGCT GGCGATGTGG ACGCCCACCA TCCCATTCAG 3480

TGTCCAGGAA CACCTGCAAC AACAGGATTC ACGGATCAGC CTGAGATGAT CCCAGCTGTG 3540

CTCTGGACAG GCATGTTCTC TGAGGACACT AACCACGCTG GACCTTGAAC TGGGTACTTG 3600

TGGACACAGC TCTTCTCCAG GCTGTATCCC ATGAGGCCTC AGCATCCTGG CACCCGGCCC 3660

CTGCTGGTTC AGGGTTGGCC CCTGCCCGGC TGCGGAATGA ACCACATCTT GCTCTGCTGA 3720

CAGACACAGG CCCGGCTCCA GGCTCCTTTA GCGCCCAGTT GGGTGGATGC CTGGTGGCAG 3780

CTGCGGTCCA CCCAGGAGCC CCGAGGCCTT CTCTGAAGGA CATTGCGGAC AGCCACGGCC 3840

AGGCCAGAGG GAGTGACAGA GGCAGCCCCA TTCTGCCTGC CCAGGCCCCT GCCACCCTGG 3900

GGAGAAAGTA CTTCTTTTTT TTTATTTTTA GACAGAGTCT CACTGTTGCC CAGGCTGGCG 3960

TGCAGTGGTG CGATCTGGGT TCACTGCAAC CTCCGCCTCT TGGGTTCAAG CGATTCTTCT 4020

›Example 7 · 3 of 15

GCTTCAGCCT CCCGAGTAGC TGGGACTACA GGCACCCACC ATCATGTCTG GCTAATTTTT 4080

CATTTTTAGT AGAGACAGGG TTTTGCCATG TTGGCCAGGC TGGTCTCAAA CTCTTGACCT 4140

CAGGTGATCC ACCCACCTCA GCCTCCCAAA GTGCTGGGGA TTACAAGCGT GAGCCACTGC 4200

ACCGGGCCAC AGAGAAAGTA CTTCTCCACC CTGCTCTCCG ACCAGACACC TTGACAGGGC 4260

ACACCGGGCA CTCAGAAGAC ACTGATGGGC AACCCCCAGC CTGCTAATTC CCCAGATTGC 4320

AACAGGCTGG GCTTCAGTGG CAGGCTGCTT TTGTCTATGG GACTCAATGC ACTGACATTG 4380

TTGGCCAAAG CCAAAGCTAG GCCTGGCCAG ATGCACCAGG CCCTTAGCAG GGAAACAGCT 4440

AATGGGACAC TAATGGGGCG GTGAGAGGGG AACAGACTGG AAGCACAGCT TCATTTCCTG 4500

TGTCTTTTTT CACTACATTA TAAATGTCTC TTTAATGTCA CAAAAAAAAA AAAAAAAAAA 4560

AAAA 4564

5105 base pairs

nucleic acid

single

linear

DNA (genomic)

cIIta gene of type IV

GGGGAGAAGT CAGAGGTAAC CTTGCCCCCT CCCTCAATTC CAGATGAGGA AATTCAGGCC 60

TGAAAAGGGA AAGTGACCAC CTCAAAGTCT CATGCCTTGG AGGACCCAGC AGGAATCCAA 120

GACCTCTGAA AAGGACCGGC AGGGCTCTTG CCACGGCTGG GGGTGTGGTC ATGGTAACAC 180

AGGTTTTCCA TCCATGGAAG GTACCTGAGG GATTTTCTCT TCCTCCCTAG GGCCAGCATC 240

AGAGGAGTGA ATAGCTCAGT TAGCTCATCT CAGGGGCCAT GTGCCCTCGG AGGTGGTTTG 300

CCACTTTCAC GGTTGGACTG AGTTGGAGAG AAACAGAGAC CCACCCAGGG GTGGGGACAA 360

GCTCCCTGCA ACTCAGGACT TGCAGATCAC TTGCCCAAGT GGCTCCCTAG CTCCTGGCTC 420

CTGGCCCGGG GCCTGGGACT CTCCCCGAAG TGGGGCTGGC CACTGTGAGG AACCGACTGG 480

AGGCAGGGAC CTCTTGGATG CCCCAGGCAG TTGGGATGCC ACTTCTGATA AAGCACGTGG 540

TGGCCACAGT AGGTGCTTGG TTGCTCCACA GCCTGGCCCG AGCTCAGCGC TGCAGAAAGA 600

AAGTGAAAGG GAAAAAGAAC TGCGGGGAGG CGGGGAGGTA GGATGACCAG CGGACGAGCT 660

GCCACAGACT TGCCGCGGCC CCAGAGCTGG CGGGAGGGAG AGGCCACCAG CAGCGCGCGC 720

GGGAGCCCGG GGAACAGCGG CAGCTCACAG TGTGCCACCA TGGAGTTGGG GCCCCTAGAA 780

GGTGGCTACC TGGAGCTTCT TAACAGCGAT GCTGACCCCC TGTGCCTCTA CCACTTCTAT 840

GACCAGATGG ACCTGGCTGG AGAAGAAGAG ATTGAGCTCT ACTCAGAACC CGACACAGAC 900

ACCATCAACT GCGACCAGTT CAGCAGGCTG TTGTGTGACA TGGAAGGTGA TGAAGAGACC 960

AGGGAGGCTT ATGCCAATAT CGCGGAACTG GACCAGTATG TCTTCCAGGA CTCCCAGCTG 1020

GAGGGCCTGA GCAAGGACAT TTTCAAGCAC ATAGGACCAG ATGAAGTGAT CGGTGAGAGT 1080

ATGGAGATGC CAGCAGAAGT TGGGCAGAAA AGTCAGAAAA GACCCTTCCC AGAGGAGCTT 1140

CCGGCAGACC TGAAGCACTG GAAGCCAGCT GAGCCCCCCA CTGTGGTGAC TGGCAGTCTC 1200

CTAGTGGGAC CAGTGAGCGA CTGCTCCACC CTGCCCTGCC TGCCACTGCC TGCGCTGTTC 1260

AACCAGGAGC CAGCCTCCGG CCAGATGCGC CTGGAGAAAA CCGACCAGAT TCCCATGCCT 1320

TTCTCCAGTT CCTCGTTGAG CTGCCTGAAT CTCCCTGAGG GACCCATCCA GTTTGTCCCC 1380

ACCATCTCCA CTCTGCCCCA TGGGCTCTGG CAAATCTCTG AGGCTGGAAC AGGGGTCTCC 1440

AGTATATTCA TCTACCATGG TGAGGTGCCC CAGGCCAGCC AAGTACCCCC TCCCAGTGGA 1500

TTCACTGTCC ACGGCCTCCC AACATCTCCA GACCGGCCAG GCTCCACCAG CCCCTTCGCT 1560

CCATCAGCCA CTGACCTGCC CAGCATGCCT GAACCTGCCC TGACCTCCCG AGCAAACATG 1620

ACAGAGCACA AGACGTCCCC CACCCAATGC CCGGCAGCTG GAGAGGTCTC CAACAAGCTT 1680

CCAAAATGGC CTGAGCCGGT GGAGCAGTTC TACCGCTCAC TGCAGGACAC GTATGGTGCC 1740

GAGCCCGCAG GCCCGGATGG CATCCTAGTG GAGGTGGATC TGGTGCAGGC CAGGCTGGAG 1800

AGGAGCAGCA GCAAGAGCCT GGAGCGGGAA CTGGCCACCC CGGACTGGGC AGAACGGCAG 1860

CTGGCCCAAG GAGGCCTGGC TGAGGTGCTG TTGGCTGCCA AGGAGCACCG GCGGCCGCGT 1920

GAGACACGAG TGATTGCTGT GCTGGGCAAA GCTGGTCAGG GCAAGAGCTA TTGGGCTGGG 1980

GCAGTGAGCC GGGCCTGGGC TTGTGGCCGG CTTCCCCAGT ACGACTTTGT CTTCTCTGTC 2040

CCCTGCCATT GCTTGAACCG TCCGGGGGAT GCCTATGGCC TGCAGGATCT GCTCTTCTCC 2100

CTGGGCCCAC AGCCACTCGT GGCGGCCGAT GAGGTTTTCA GCCACATCTT GAAGAGACCT 2160

GACCGCGTTC TGCTCATCCT AGACGCCTTC GAGGAGCTGG AAGCGCAAGA TGGCTTCCTG 2220

CACAGCACGT GCGGACCGGC ACCGGCGGAG CCCTGCTCCC TCCGGGGGCT GCTGGCCGGC 2280

CTTTTCCAGA AGAAGCTGCT CCGAGGTTGC ACCCTCCTCC TCACAGCCCG GCCCCGGGGC 2340

CGCCTGGTCC AGAGCCTGAG CAAGGCCGAC GCCCTATTTG AGCTGTCCGG CTTCTCCATG 2400

GAGCAGGCCC AGGCATACGT GATGCGCTAC TTTGAGAGCT CAGGGATGAC AGAGCACCAA 2460

GACAGAGCCC TGACGCTCCT CCGGGACCGG CCACTTCTTC TCAGTCACAG CCACAGCCCT 2520

ACTTTGTGCC GGGCAGTGTG CCAGCTCTCA GAGGCCCTGC TGGAGCTTGG GGAGGACGCC 2580

AAGCTGCCCT CCACGCTCAC GGGACTCTAT GTCGGCCTGC TGGGCCGTGC AGCCCTCGAC 2640

AGCCCCCCCG GGGCCCTGGC AGAGCTGGCC AAGCTGGCCT GGGAGCTGGG CCGCAGACAT 2700

CAAAGTACCC TACAGGAGGA CCAGTTCCCA TCCGCAGACG TGAGGACCTG GGCGATGGCC 2760

AAAGGCTTAG TCCAACACCC ACCGCGGGCC GCAGAGTCCG AGCTGGCCTT CCCCAGCTTC 2820

CTCCTGCAAT GCTTCCTGGG GGCCCTGTGG CTGGCTCTGA GTGGCGAAAT CAAGGACAAG 2880

GAGCTCCCGC AGTACCTAGC ATTGACCCCA AGGAAGAAGA GGCCCTATGA CAACTGGCTG 2940

GAGGGCGTGC CACGCTTTCT GGCTGGGCTG ATCTTCCAGC CTCCCGCCCG CTGCCTGGGA 3000

GCCCTACTCG GGCCATCGGC GGCTGCCTCG GTGGACAGGA AGCAGAAGGT GCTTGCGAGG 3060

TACCTGAAGC GGCTGCAGCC GGGGACACTG CGGGCGCGGC AGCTGCTTGA GCTGCTGCAC 3120

TGCGCCCACG AGGCCGAGGA GGCTGGAATT TGGCAGCACG TGGTACAGGA GCTCCCCGGC 3180

CGCCTCTCTT TTCTGGGCAC CCGCCTCACG CCTCCTGATG CACATGTACT GGGCAAGGCC 3240

TTGGAGGCGG CGGGCCAAGA CTTCTCCCTG GACCTCCGCA GCACTGGCAT TTGCCCCTCT 3300

GGATTGGGGA GCCTCGTGGG ACTCAGCTGT GTCACCCGTT TCAGGGCTGC CTTGAGCGAC 3360

ACGGTGGCGC TGTGGGAGTC CCTGCGGCAG CATGGGGAGA CCAAGCTACT TCAGGCAGCA 3420

GAGGAGAAGT TCACCATCGA GCCTTTCAAA GCCAAGTCCC TGAAGGATGT GGAAGACCTG 3480

GGAAAGCTTG TGCAGACTCA GAGGACGAGA AGTTCCTCGG AAGACACAGC TGGGGAGCTC 3540

CCTGCTGTTC GGGACCTAAA GAAACTGGAG TTTGCGCTGG GCCCTGTCTC AGGCCCCCAG 3600

GCTTTCCCCA AACTGGTGCG GATCCTCACG GCCTTTTCCT CCCTGCAGCA TCTGGACCTG 3660

GATGCGCTGA GTGAGAACAA GATCGGGGAC GAGGGTGTCT CGCAGCTCTC AGCCACCTTC 3720

CCCCAGCTGA AGTCCTTGGA AACCCTCAAT CTGTCCCAGA ACAACATCAC TGACCTGGGT 3780

GCCTACAAAC TCGCCGAGGC CCTGCCTTCG CTCGCTGCAT CCCTGCTCAG GCTAAGCTTG 3840

TACAATAACT GCATCTGCGA CGTGGGAGCC GAGAGCTTGG CTCGTGTGCT TCCGGACATG 3900

GTGTCCCTCC GGGTGATGGA CGCAAGTTCA CGGCTGCCGG GGCCCAGCAG CTCGCTGCCA 3960

GCCTTCGGAG GTGTCCTCAT GTGGAGACGC TGGCGATGTG GACGCCCACC ATCCCATTCA 4020

GTGTCCAGGA ACACCTGCAA CAACAGGATT CACGGATCAG CCTGAGATGA TCCCAGCTGT 4080

GCTCTGGACA GGCATGTTCT CTGAGGACAC TAACCACGCT GGACCTTGAA CTGGGTACTT 4140

GTGGACACAG CTCTTCTCCA GGCTGTATCC CATGAGGCCT CAGCATCCTG GCACCCGGCC 4200

CCTGCTGGTT CAGGGTTGGC CCCTGCCCGG CTGCGGAATG AACCACATCT TGCTCTGCTG 4260

ACAGACACAG GCCCGGCTCC AGGCTCCTTT AGCGCCCAGT TGGGTGGATG CCTGGTGGCA 4320

GCTGCGGTCC ACCCAGGAGC CCCGAGGCCT TCTCTGAAGG ACATTGCGGA CAGCCACGGC 4380

CAGGCCAGAG GGAGTGACAG AGGCAGCCCC ATTCTGCCTG CCCAGGCCCC TGCCACCCTG 4440

GGGAGAAAGT ACTTCTTTTT TTTTATTTTT AGACAGAGTC TCACTGTTGC CCAGGCTGGC 4500

GTGCAGTGGT GCGATCTGGG TTCACTGCAA CCTCCGCCTC TTGGGTTCAA GCGATTCTTC 4560

›Example 7 · 4 of 15

TGCTTCAGCC TCCCGAGTAG CTGGGACTAC AGGCACCCAC CATCATGTCT GGCTAATTTT 4620

TCATTTTTAG TAGAGACAGG GTTTTGCCAT GTTGGCCAGG CTGGTCTCAA ACTCTTGACC 4680

TCAGGTGATC CACCCACCTC AGCCTCCCAA AGTGCTGGGG ATTACAAGCG TGAGCCACTG 4740

CACCGGGCCA CAGAGAAAGT ACTTCTCCAC CCTGCTCTCC GACCAGACAC CTTGACAGGG 4800

CACACCGGGC ACTCAGAAGA CACTGATGGG CAACCCCCAG CCTGCTAATT CCCCAGATTG 4860

CAACAGGCTG GGCTTCAGTG GCAGGCTGCT TTTGTCTATG GGACTCAATG CACTGACATT 4920

GTTGGCCAAA GCCAAAGCTA GGCCTGGCCA GATGCACCAG GCCCTTAGCA GGGAAACAGC 4980

TAATGGGACA CTAATGGGGC GGTGAGAGGG GAACAGACTG GAAGCACAGC TTCATTTCCT 5040

GTGTCTTTTT TCACTACATT ATAAATGTCT CTTTAATGTC ACAAAAAAAA AAAAAAAAAA 5100

AAAAA 5105

717 base pairs

nucleic acid

single

linear

DNA (genomic)

cIIta promoter of type I